Targeting of Sodium Channel Blockers Into Nociceptors to Produce

British Journal of DOI:10.1111/j.1476-5381.2011.01391.x www.brjpharmacol.org BJP Pharmacology

REVIEWbph_1391 48..58 Correspondence Clifford J Woolf, F.M. Kirby Neurobiology Center, Children’s Hospital Boston, 300 Longwood Targeting of sodium channel Avenue, CLS 12258, Boston, MA 02115, USA. E-mail: [email protected] blockers into nociceptors to ------*These authors contributed equally to this work as ﬁrst produce long-duration author. ------Keywords analgesia: a systematic QX-314; TRPV1 channels; lidocaine; sodium channel blockers; long-duration analgesia; study and review differential block; regional anaesthesia; nerve block; targeted

1,2,3 1,4 1 2 1,2 delivery DP Roberson *, AM Binshtok *, F Blasl , BP Bean and CJ Woolf ------Received 1FM Kirby Neurobiology Center and Department of Neurology, Children’s Hospital, Boston, MA, 31 August 2010 2 3 USA, Department of Neurobiology, Harvard Medical School, Boston, MA, USA, Rappaport Revised Faculty of Medicine, Technion American Medical School, Technion Israel Institute of Technology, 2 March 2011 Haifa, Israel, and 4Department of Medical Neurobiology, Institute for Medical Research Accepted Israel-Canada, The Hebrew University Medical School, Jerusalem, Israel 7 March 2011

BACKGROUND AND PURPOSE We have developed a strategy to target the permanently charged lidocaine derivative lidocaine N-ethyl bromide (QX-314) selectively into nociceptive sensory neurons through the large-pore transient receptor potential cation channel subfamily V (TRPV1) noxious heat detector channel. This involves co-administration of QX-314 and a TRPV1 agonist to produce a long-lasting local analgesia. For potential clinical use we propose using lidocaine as the TRPV1 agonist, because it activates TRPV1 at clinical doses. EXPERIMENTAL APPROACH We conducted experiments in rats to determine optimal concentrations and ratios of lidocaine and QX-314 that produce the greatest degree and duration of pain-selective block when administered nearby the sciatic nerve: reduction in the response to noxious mechanical (pinch) and to radiant heat stimuli, with minimal disruption in motor function (grip strength). KEY RESULTS A combination of 0.5% QX-314 and 2% lidocaine produced 1 h of non-selective sensory and motor block followed by >9hof pain-selective block, where grip strength was unimpaired. QX-314 at this concentration had no effect by itself, while 2% lidocaine by itself produced 1 h of non-selective block. The combination of 0.5% QX-314 and 2% lidocaine was the best of the many tested, in terms of the duration and selectivity of local analgesia. CONCLUSIONS AND IMPLICATIONS Targeting charged sodium channel blockers into specific sets of axons via activation of differentially expressed large-pore channels provides an opportunity to produce prolonged local analgesia, and represents an example of how exploiting ion channels as a drug delivery port can be used to increase the specificity and efficacy of therapeutics.

Abbreviation QX-314, lidocaine N-ethyl bromide

Introduction derivatives of local anaesthetics have no effect if applied externally but have blocking activity if applied on the cyto- The most powerful known method of blocking pain while plasmic side of the membrane, as first shown using lidocaine retaining consciousness is to inject local anaesthetics like N-ethyl bromide (QX-314), a lidocaine derivative with a per- lidocaine regionally into areas of the body generating pain. manent positive charge conferred by a quaternary nitrogen Lidocaine produces its local aesthetic actions by blocking (Frazier et al., 1970; Strichartz, 1973). Lidocaine itself has a voltage-gated sodium channels. Like all local anaesthetics, tertiary nitrogen with pKa of 8.2, so that a pH of 7.4 ~ 15% of lidocaine has little or no selectivity among different types of the molecules will be in the unprotonated, uncharged state, sodium channels (Hille, 1977a; Schwarz et al., 1977; Liu et al., which is highly permeable and provides rapid entry into the 2003; Chevrier et al., 2004; Leffler et al., 2005) and its local cell (Hille, 1977b). Once inside, protonation occurs to estab- aesthetic action is also non-selective, blocking action poten- lish charged as well as uncharged forms of the molecule. It is tials in all sensory, motor and autonomic fibres. In particular, likely that both charged and uncharged forms of the drug can it blocks both low threshold sensory axons carrying innocu- bind and block the channels from the cytoplasmic surface, ous information and high threshold (nociceptor) axons that because benzocaine, an uncharged molecule similar to the contribute to painful sensations. The net effect of a lidocaine uncharged form of lidocaine, blocks sodium channels nearly injection close to a nerve at a therapeutically effective dose (1 as potently as does lidocaine (Hille, 1977a,b; Schwarz et al., to 2%, 35 to 69 mM) (Enneking et al., 2009), is complete 1977; Clapham et al., 2001). sensory and motor block, including loss of all sensation The ability of QX-314 to block from the inside but not the (numbness), paralysis and abolition of autonomic function. outside of neuronal membranes could be exploited to block While such an outcome may be acceptable in some settings, only selected neurons if there were some way to allow it to such as during surgery, there are many clinical situations enter some neurons but not others. A possible strategy to do where a selective block of some but not other axons would be this is to use naturally expressed large-pore ion channels as an more desirable. For example, block of nociceptors to produce entry port for QX-314 (or similar permanently charged analgesia without a loss of proprioception or motor function sodium channel blockers) into neurons. The candidate would enable early mobilization in patients receiving periph- channel we chose to investigate first was transient receptor eral nerve block or plexus block, for example, following knee potential cation channel subfamily V (TRPV1), a member of or hip joint replacement. the large transient receptor transient receptor potential (TRP) A further issue with regional injections of local anaesthet- channel family (Clapham et al., 2001). The reason for this was ics is their relative short duration, limited to several hours, twofold. First, the channel has been shown to permeate large which is usually not enough to fully cover the normal dura- cations such as tetraethylammonium (130 Da) and N-methyl- tion of post-operative pain. Furthermore, because of D-glucamine (195 Da) (Hellwig et al., 2004; Oseguera et al., lidocaine’s action on central neurons and cardiac muscle, it 2007) and surprisingly, even a very large cationic dye FM1-43 can have major central nervous system and cardiovascular (452 Da) (Meyers et al., 2003) which, together with TRPV1’s toxicity problems when administered locally at high volumes high single-channel conductance (Premkumar et al., 2002; (Dillane and Finucane, 2010; Neal et al., 2010). There is there- Raisinghani et al., 2005), suggests that the channel has a fore a need for a pharmacological therapy that has more large-pore, certainly large enough to permeate cationic drugs selectivity for nociceptors, a longer duration and a reduced like QX-314 (263 Da). Activation of native or recombinant side effect burden. TRPV1 also leads to time- and agonist concentration- How can a selective block of nociceptors be achieved to dependent increases in permeability to large cations like produce a local analgesia instead of a non-specific local ana- N-methyl-D-glucamine (NMDG+, 195 Da) (Chung et al., esthesia? One way would be to selectively target those 2008). Such pore dilation also occurs for transient receptor voltage-gated sodium channels expressed only or predomi- potential subfamily A1 (TRPA1) but not transient receptor nantly in these neurons, such as Nav1.7, 1.8 and 1.9 (Wood potential M8 (Chen et al., 2009). The second reason, we et al., 2004; Priest, 2009; see also Momin and Wood, 2008; looked at TRPV1 is because it is a noxious heat detector Dib-Hajj et al., 2009). However, only a few subtype selective (Caterina et al., 1997; Premkumar and Ahern, 2000), and is sodium channel blockers have been reported (Priest, 2009; therefore almost exclusively expressed in nociceptors. Thus, if Zhang et al., 2010), and none have been shown to produce we could selectively use TRPV1 to permeate QX-314 into local analgesia. neurons we could potentially achieve a pain specific block. We have developed an alternative strategy, one based on The first way we examined this hypothesis was to use a targeting a sodium channel blocker to selectively enter into combination of QX-314 and capsaicin, a TRPV1 agonist and nociceptors and not into low threshold sensory and motor the pungent ingredient in chilli peppers (Binshtok et al., axons. This strategy is based on the location of the binding 2007). We found that QX-314, when administered alone to site of local anaesthetics on the inside of the pore of sodium dorsal root ganglion neurons, was without effect on voltage- channels, in a region involving the pore-lining S6 regions of gated sodium current, as expected. In contrast, co-application the pseudo-subunit domains I, III and IV of the channel of QX-314 with capsaicin dramatically inhibited sodium (Ragsdale et al., 1994; 1996; Yarov-Yarovoy et al., 2002; current (by ~90%), consistent with QX-314 entering the McNulty et al., 2007; Sheets and Hanck, 2007; Ahern et al., neurons through TRPV1 channels and blocking from the 2008; Muroi and Chanda, 2009). For neuronal sodium chan- inside. This action completely abolished the ability to gener- nels, local aesthetic molecules can apparently access the ate action potentials and was seen only in small TRPV1 binding site only from the cytoplasmic end of the pore, expressing dorsal root ganglion (DRG) neurons, with large because application of charged, membrane-impermeant non-capsaicin-responsive neurons unaffected (Binshtok et al.,

British Journal of Pharmacology (2011) 164 48–58 49 BJP DP Roberson et al.

2007). The effect was also seen in TRPV1-expressing trigemi- entry of QX-314 into nociceptors: both lidocaine and QX-314 nal ganglion neurons, where it was also shown that block of are water soluble so there are no formulation issues, lidocaine sodium current and action potentials is irreversible after has already been studied extensively for toxicology, and as washing capsaicin and QX-314, consistent with QX-314 QX-314 is a simple derivative of lidocaine, its toxicology being trapped inside the neurons after TRPV1 channels close might be expected to be generally similar. However, because of (Kim et al., 2010). lidocaine’s actions as both an indiscriminate blocker of all In vivo experiments suggested that TRPV1-mediated entry excitability and as a TRPV1 agonist, it is clear that a key issue of QX-314 can be used to produce nociceptor-selective block in the potential clinical use of the combination of lidocaine of excitability and axonal conduction. Local injection in and QX-314 is to determine optimal concentrations of the rodents of QX-314 alone was, as expected, without effect two molecules to produce long-lasting nociceptor block while (Binshtok et al., 2007; 2009a). Injection of capsaicin alone minimizing the duration of motor block. A further concern is subcutaneously elicited a nociceptive reaction that lasted to determine whether this can be done with total concentra- about ~15 min (Binshtok et al., 2007) and a similar reaction tions of both drugs at a level likely to be acceptable from a was elicited by perineural injection (Binshtok et al., 2009a), toxicological standpoint. To address these issues, we have reflecting the presence of TRPV1 expression on the axons of conducted a study, reported below, testing a range of concen- nociceptors in peripheral nerves (Hoffmann et al., 2008). trations of both agents for producing prolonged local analge- However, when QX-314 was co-applied with capsaicin, either sia while minimizing motor block. subcutaneously or perineurally, there was a long-lasting block of heat and mechanical pain, with no block in motor function (Binshtok et al., 2007). Subsequent experiments on the Methods jaw opening reflex confirmed the specificity of the combination for nociceptor fibres in sensory nerves, and demon- Animal procedures were approved by the Committee on strated blockade of dental pain (Kim et al., 2010). Research Animal Care of the Massachusetts General Hospital, We interpreted these data as showing that we could Boston, MA. Male Sprague-Dawley rats were purchased from indeed exploit TRPV1 as a ‘drug-delivery portal’ mechanism Charles River Laboratories, Inc., Wilmington, MA, USA. The to target QX-314 into neurons at sufficient concentrations to rats were habituated to handling and experimental proce- block sodium currents and action potentials, with the differ- dures for 1 week prior to testing. At the time of injection, rats ential expression of TRPV1 providing specificity for delivery were approximately 6.5 weeks old and weighed approxi- of the drug only into nociceptors. The long duration of the mately 200–250 g. Each of the experiments utilized concur- effect presumably reflects trapping of QX-314 in the axon, rent observation of a mixed cohort of three test groups where unlike lidocaine it cannot diffuse out the membrane (groups n = 9, cohort n = 27), with the experimenter blind to and will either diffuse along the axon, or slowly be removed the treatments. by exocytosis, degradation or slow leakage through channels. QX-314 bromide salt (Cat. No. L5783, Sigma, St. Louis, While our strategy had been shown to work, there MO, USA) and lidocaine hydrochloride monohydrate (Cat. remained an important problem for its clinical exploitation. No. L5647, Sigma, St. Louis, MO, USA) were prepared freshly Activation of TRPV1 channels by capsaicin occurs instantly in normal saline (0.9% NaCl, 200 mL; Sigma, St. Louis, MO, (<1 s), while entry of enough QX-314 to block action poten- USA) to the predetermined concentrations (percent weight by tials takes several minutes (Binshtok et al., 2007). This delay is volume) immediately prior to injection. The pH of tested long enough for the capsaicin administration to produce solutions ranged from 5.0 to 6.3 and was not adjusted due to several minutes of high-level nociceptor activation, which in the probability of rapid buffering by the pH of the extracel- humans would elicit severe burning pain (Gustafsson et al., lular fluid within tissue. 2009), only after which, the long-lasting pain-selective block would manifest. How to overcome this? One solution would be use non-pungent agonists of TRPV1, like eugenol (Yang Sciatic nerve injections et al., 2003), which is the active ingredient in oil of cloves. Rats were lightly anaesthetized by inhalation of isoflurane While we found that a combination of QX-314 and eugenol (1.5–2%, in oxygen) for approximately 5–7 min, and the could indeed reduce sodium currents in vitro, formulation landmarks (greater trochanter and ischial tuberosity) of the problems prevented co-application in vivo. Fortuitously, left hind limb localized. Groups of six rats were injected with however, a concurrent study by Andreas Leffler and colleagues 0.2 mL of each test solution: lidocaine (1%, 1.5%, 2%), revealed the remarkable fact that lidocaine itself, at clinically QX-314 (0.25%, 0.5%, 1%) and lidocaine mixed with QX-314 administered concentrations (30 mM), is a TRPV1 agonist. (1% lidocaine + 0.25% QX-314, 1% lidocaine + 0.5% QX-314, They showed that lidocaine produced calcium influx in DRG 1% lidocaine + 1% QX-314, 1.5% lidocaine + 0.5% QX- neurons that was blocked by a TRPV1 antagonist and could 314, 2% lidocaine + 0.5% QX-314, 2% lidocaine + 1% QX- activate heterologously expressed TRPV1 channels (Leffler 314). The drug was injected in immediate proximity to the et al., 2008). This led us to test if we could substitute lidocaine sciatic nerve with a 27-gauge hypodermic needle attached to for capsaicin as a TRPV1 agonist for in vivo experiments. The a tuberculin syringe. For the experiments described in results were promising, with a combination of lidocaine and Figure 4, QX-314 (1%) and vehicle were injected to unanaes- QX-314 producing much longer analgesia than lidocaine thetized rats. The animals (n = 18) were manually restrained alone (Binshtok et al., 2009a). In principle, the combination and sciatic injections performed as described above. of lidocaine and QX-314 seems an ideal method for develop- Two baseline readings of each test modality were taken; ment of a clinical therapy using TRPV1 channels to target one at 24 h prior to injection and another immediately prior

50 British Journal of Pharmacology (2011) 164 48–58 Targeting sodium channel blockers for analgesia BJP to induction of isoﬂurane general anaesthesia (approximately normalized at each time point by subtracting the group mean

10 min prior to injection). Additional measurements were behavioural response score at baseline (xt = 0) from the group recorded at 30 min, 60 min, and 4, 6, 9, 12 and 24 h after behavioural response score (x) and dividing by the behav- injection. Observer variability was determined to be insigniﬁ- ioural response cut-off (xcut-off) minus group mean behavioural cant by comparison of data obtained during the training response score at baseline (xt = 0) as described in the following period (n = 18). equation:

Normalized behavioural response score Motor function =−(xxtcut-offt=0 )( x − x=0 ) The Bioseb Grip Strength Test apparatus was used to assess changes in grasping strength of the left hind limb according to the method described by Simon et al. (Simon et al., 2004). Normal response in untreated rats ~200 g, while the response during a complete lidocaine 2% block was ~5g. Results

We have previously demonstrated that the combined appli- Sensory function cation of QX-314 together with lidocaine (lidocaine HCl) Ugo Basile model no. 7371 was used to assess changes in produces a prolonged nociceptive-selective blockade, which thermal nociceptive response latency upon application of follows the brief non-selective effects of lidocaine (Binshtok 52°C radiant heat at the lateral plantar surface of left hind et al., 2009a). We determined that perisciatic injection of a paw according to the method described by Hargreaves et al. fixed 0.2% concentration of QX-314 together with different (Hargreaves et al., 1988). Normal response ~16 s, cut-off 25 s. concentrations of lidocaine (0.5, 1, 2%) blocked the nocifen- The Bioseb Rodent Pincher Analgesia Meter was used to sive response to pinch, an effect that persisted well beyond assess changes in mechanical nociception elicited upon the duration of the transient motor block, as measured by the pinch of the fifth proximal phalanx of the left hind paw, extensor postural thrust test. The duration of the differential according to the method described by Luis-Delgado et al. block was increasingly prolonged with higher concentrations (Luis-Delgado et al., 2006). Normal responses approximated of lidocaine (Binshtok et al., 2009a). 200 g in each group. Cut-off was set at 500 g and was Here, we hypothesized that by modifying the dose-ratio achieved within 5 s in all animals. No damage to skin or deep of QX-314 and lidocaine we could further prolong the dura- tissue was evident at cut-off level. tion of the nociceptive selective block over the motor block and thereby optimize the duration of nociceptive-specific Statistical analysis differential block for potential clinical use. To test this we Data is presented as mean Ϯ SEM. Analysis of injections was applied different dose combinations of both QX-314 and done with either one-way analysis of variance (ANOVA) fol- lidocaine close to the sciatic nerve of adult rats and assessed lowed by Dunnett’s test (compared with baseline values) or the changes in nociceptive threshold and motor strength at two-way ANOVA followed by Bonferroni (comparison between different time points after injection, to ascertain the particu- different treatments). To evaluate the significance of differ- lar dose combination producing an optimal duration of dif- ences in the area under the curve (AUC) ANOVA with post hoc ferential block. Dunnett’s test were performed. AUC were calculated as the Perisciatic injection of 1% lidocaine (200 mL) alone pro- trapezoidal area under the behavioural response score versus duced a short-lasting blockade of the response to noxious time curve for each behavioural modality (grip strength, mechanical (pinch) and thermal (radiant heat) sensation that pinch tolerance or thermal response latency). Scores were was no longer significant after 30 min (P < 0.01) (Figure 1A

Figure 1 The duration of the elevation in thermal response latency (radiant heat, 50°C), pinch tolerance threshold and reduction in grip strength produced following perisciatic injection of lidocaine HCl (1%, 1.5%, 2%) and vehicle (0.9% NaCl) (mean Ϯ SEM; *P < 0.01). All injections administered under isoﬂurane-induced general anaesthesia at time 0.

British Journal of Pharmacology (2011) 164 48–58 51 BJP DP Roberson et al.

Figure 2 The duration of the elevation in thermal (radiant heat, 50°C) response latency (A, D and G), pinch tolerance threshold (B, E and H), and reduction in grip strength (C, F and I) produced following perisciatic (200 mL) injection of lidocaine alone (1%, 1.5% or 2%), 0.5% lidocaine N-ethyl bromide (QX-314) alone, or 0.5% QX-314 administered together with lidocaine (1%, 1.5% or 2%) (mean Ϯ SEM; *P < 0.01). All injections administered under isoﬂurane-induced general anaesthesia at time 0.

and B). The duration of motor block was coincident with the Injection of vehicle (0.9% NaCl) did not cause any sig- nociceptive block, lasting 30 min (Figure 1C) (P < 0.01). An niﬁcant changes in motor and nociceptive function increase in the concentration of lidocaine to 1.5% prolonged (Figure 1). Similarly, injection of 0.5% QX-314 alone did not the duration of blockade to noxious mechanical stimuli up to change the responses to noxious mechanical and thermal 1 h, without further prolonging the duration of the blockade stimuli, and did not produce motor weakness (reduced grip of response to noxious thermal stimuli or motor block. strength) (Figure 2). Perineural injection of 2% lidocaine alone produced sensory When combined with lidocaine, QX-314 prolonged the and motor block for 1 h (Figure 1). Thus, consistent with our duration of both sensory and motor block for all doses of previous observations, application of lidocaine in therapeu- lidocaine tested. All examined combinations of lidocaine and tically relevant doses did not produce any differential block QX-314 resulted in a sensory block that exceeded the dura- and its effect was relatively short lasting. tion of the motor block. The duration of the differential

52 British Journal of Pharmacology (2011) 164 48–58 Targeting sodium channel blockers for analgesia BJP block, however, varied according to the dose of both lidocaine and QX-314. Co-application of 0.5% QX-314 with 1% lidocaine produced a blockade of the responses to noxious thermal and mechanical stimuli that was significant for 6 h (P < 0.01), and a motor block that was significant for 4 h (P < 0.01), resulting in 2 h of differential block (Figure 2A–C). Injection of 1.5% of lidocaine together with 0.5% QX-314 further prolonged the blockade of the response to noxious mechanical stimuli to 9h (P < 0.01) (Figure 2E). The duration of the blockade of response to noxious thermal stimuli was prolonged to 6 h (P < 0.01) (Figure 2E), while the duration of the motor block remained similar to that produced by the combination of 1% lidocaine and 0.5% QX-314 (Figure 2C and F). Increasing the concentration of lidocaine to 2%, while maintaining QX-314 concentration at 0.5%, prolonged the duration of blockade to noxious thermal and mechanical stimuli to 9 h (P < 0.01), thus inducing a nociceptive block that persisted 8 h beyond the blockade produced by the injection of 2% lidocaine alone (Figure 2G and H). Surprisingly, the duration of motor block resulting from injection of 2% lidocaine together with 0.5% QX-314 lasted only 1 h longer Figure 3 than the motor block induced by 2% lidocaine alone Analysis of the change in grip strength, thermal (radiant heat, 50°C) (Figure 2I). The duration of this motor block was considerably response latency and pinch tolerance threshold produced following shorter than the motor block produced by corresponding perisciatic injection of varying doses of lidocaine N-ethyl bromide combinations containing lower concentrations of lidocaine (QX-314) (0%, 0.5%) and lidocaine (0%, 1%, 2%) expressed as total area under the curve (AUC). Note that the value of AUC representing (Figures 1 and 2C, F and I). change in pinch tolerance threshold in the group receiving a com- The combination of 0.5% QX-314 (which has no signifi- bined dose of 0.5% QX-314 + 2% lidocaine, is greater than the cant effect when administered on its own) with 2% lidocaine combined values of corresponding AUCs from the group receiving (which has a brief non-selective action when administered on 0.5% QX-314 alone plus the AUC from the group receiving 2.0% its own) produces a long-lasting decrease in pinch sensitivity lidocaine alone. Similarly, the AUC value representing change in (pinch) and noxious thermal (radiant heat) responsiveness. thermal latency in the group receiving a combined dose of 0.5% Addition of 0.5% QX-314 to 2% lidocaine has a minimal QX-314 + 2% lidocaine, is much greater than the combined values of effect on grip strength versus 2% lidocaine alone. AUC analy- corresponding AUCs from the group receiving 0.5% QX-314 alone sis demonstrated that the effect of this particular combina- plus the AUC from the group receiving 2.0% lidocaine alone. Con- tion of lidocaine and QX-314 on radiant heat response versely, the value of AUC representing grip strength in the group receiving a combined dose of 0.5% QX-314 + 2% lidocaine, is less latency [(see Methods for the details of the normalization than the combined values of grip strength AUCs from the group method) normalized AUC (nAUC) Lidocaine 2% + QX-314 0.5% = 8; receiving 0.5% QX-314 alone plus the grip strength AUC from the nAUC lidocaine 2% = 1.1; nAUC QX-314 0.5% = 0.23] and pinch toler- group receiving 2.0% lidocaine alone. ance (nAUC Lidocaine 2% + QX-314 0.5% = 9.2; nAUC lidocaine 2% = 1.4; nAUC QX-314 0.5% = 0.3) is much greater than the additive effects of the two drugs administered individually, but the pinch), but also prolonged the motor block to 6 h (P < 0.01) effect on the grip strength (nAUC Lidocaine 2% + QX-314 0.5% = 2.1; (Figure S1). Injection of 2% lidocaine and 1% QX-314 pro- nAUC lidocaine 2% = 1.7; nAUC QX-314 0.5% = 1.7) is less than the duced 12 h of sensory block (P < 0.01) and 9 h of motor block additive effects of the two drugs administered individually (P < 0.01) (data not shown). (Figure 3). Surprisingly, application of 1% QX-314 alone (i.e. without Because the optimal lidocaine concentration for sciatic lidocaine) produced a differential sensory block characterized nerve block is similar between rat and humans (Nakamura by a reduction of noxious mechanical threshold persisting for et al., 2003), and in order to limit potential lidocaine toxicity 12 h (P < 0.05) and a blockade of the response to noxious that may arise from addition of a second lidocaine-based thermal stimuli lasting for 6 h (P < 0.01). The injected agent such as QX-314, we did not exceed the clinically rec- animals also demonstrated a motor weakness that continued ommended concentration range (1–2%) for optimal single- for 2 h (P < 0.05) (Figure 4). Because the present experiments injection sciatic nerve block in humans (Enneking et al., were all performed under isoflurane-induced general anaes- 2009). We thus decided to increase the concentration of thesia to facilitate perisciatic nerve injections, we hypoth- QX-314 in combination with clinically relevant doses of esized that the isoflurane-mediated activation of TRPV1 lidocaine (1%, 1.5%, 2%). Increasing the concentration and/or TRPA1 (Harrison and Nau, 2008; Matta et al., 2008) of QX-314 from 0.5% to 1% did not further enhance the may permit QX-314 entry into nociceptors at QX-314 con- duration of differential block. Specifically, the application of centrations greater than or equal to 1%. 1% lidocaine together with 1% QX-314 prolonged the dura- To determine whether the appearance of a non-selective tion of thermal nociceptive block to 9 h (radiant heat; block by high doses of QX-314 administered on its own was P < 0.01) and mechanical nociceptive block to 12 h (P < 0.01; a consequence of the isoflurane general anaesthesia, we con-

British Journal of Pharmacology (2011) 164 48–58 53 BJP DP Roberson et al.

Figure 4 The motor and sensory block following injection of 1% lidocaine N-ethyl bromide (QX-314) is abolished when injected in the absence of general anaesthesia. Perisciatic application of 1% QX-314 alone produces prolonged elevation in thermal (radiant heat, 50°C) response latency (A), pinch tolerance threshold (B) and grip weakness (C) only while applied under isoﬂurane-induced general anaesthesia. Perisciatic injection of 1% QX-314 in non-anaesthetized animals did not change the responses to noxious mechanical and thermal stimuli or grip force. Application of vehicle (0.9% NaCl) administered without general anaesthesia also did not alter motor, mechanical or thermal responsiveness. Values expressed as percent of maximal block (mean Ϯ SEM; *P < 0.01, †P < 0.01, ANOVA followed by Dunnett’s test; n = 9 for each group). All injections administered at time 0.

ducted a series of experiments where the perisciatic injection and Tsui, 2010; Power et al., 2010), the non-selective action of of QX-314 (1%) was performed in the absence of isoflurane currently available sodium channel blockers means that a general anaesthesia. The sensory and motor blocking effects block of motor, sensory and autonomic function inevitably of 1% QX-314 administered alone in the presence of isoflu- occurs, even if only analgesia is required. Our strategy of rane were totally abolished in the absence of general anaes- using large-pore channels to deliver sodium channel blockers thesia (Figure 4), indicating that isoflurane can induce a into nociceptors (Binshtok et al., 2007) provides an alterna- means of entry for high concentrations of QX-314 into tive approach. In its ideal form, this approach incorporates axons. The sensory blockade produced by QX-314 under both a TRPV1 agonist and a permanently charged sodium general anaesthesia at concentrations exceeding 1% suggests channel blocker such as QX-314 to produce a block only of that isoflurane mediated activation of TRPV1 and/or TRPA1 nociceptors (Binshtok et al., 2007). However, patients would may provide a passage for QX-314 into nociceptors. However, simply not tolerate the initial pain that would be produced by QX-314 alone at high doses in the presence of isoflurane also injection of a TRPV1 agonist like capsaicin prior to produc- produced a motor block implying some action on channels tion of the nociceptor block. expressed by motor axons. While the results of such non- As an alternative strategy, we have chosen to activate anaesthetized groups are of obvious mechanistic interest, the TRPV1 using lidocaine because its activation of TRPV1 chan- stress induced by conscious perisciatic injections, requiring nels (Leffler et al., 2008) – although substantial at clinically restraint, together with lack of a clinical correlate, convinced used doses (>5 mM) – is masked within seconds by its sodium us that broader studies of perisciatic injections in absence of channel blocking action so that only a very transient burning general anaesthesia were not warranted, as our prime effort sensation is experienced (Davies, 2003; Vossinakis et al., was focused on finding maximal differential block even when 2004). While co-administration of lidocaine with QX-314 can administered under general anaesthesia, for potential clinical target QX-314 through TRPV1 into nociceptor neurons in exploitation. We conclude therefore, that a combination of culture (unpublished observations), this is obviously at the 0.5% QX-314 and 2% lidocaine is the optimal concentration expense of an initial period of non-selective block (Binshtok and ratio for producing the longest-duration differential et al., 2009a), as demonstrated by the short-lasting reduction block. in grip strength in the current experiments. However, the early non-selective block produced by the lidocaine is followed by a much longer period of differential block due to the Discussion and conclusions distribution of QX-314 into nociceptors, where the response to noxious mechanical and thermal stimuli is very substan- Regional anaesthesia with local anaesthetic agents has the tially reduced, even after motor function has fully recovered. great advantage over general anaesthesia of targeting treat- This profile of short non-selective block followed by a pro- ment to the affected site, whether by local tissue/perineural longed pain-selective block produced by the lidocaine/QX- injection or epidural/intrathecal delivery, thus avoiding or 314 combination may have utility for many surgical minimizing systemic side effects. Although very successful for procedures. For example, the initial non-selective block many surgical interventions (Hogan et al., 2009; Fredrickson would be advantageous during surgery, while the longer- et al., 2010; Hawkins, 2010; Murray et al., 2010; Scott, 2010) lasting local analgesia would be beneficial during the post- as well treatment of some chronic pain conditions (Dillane surgical period; a long-lasting effect that is absent when

54 British Journal of Pharmacology (2011) 164 48–58 Targeting sodium channel blockers for analgesia BJP lidocaine is administered alone. Clinically, such long-lasting nerve block (Enneking et al., 2009). We therefore decided that local post-operative analgesia with intact motor function 2% lidocaine (69 mM) would be the maximal dose used in could contribute to more rapid mobilization and decreased the present study. Leffler et al. demonstrated that lidocaine, at requirements for intra/post-operative opioids, both of which this concentration, also activates the TRPA1 channel – would be valuable to patients and caregivers, particularly in another nociceptive specific transducer that involved in an outpatient surgical setting, because it could allow earlier detection of noxious cold and various harmful chemicals hospital discharge and better pain control. More generally, (Leffler et al., 2008). We recently demonstrated that the the inherent advantages of early mobilization would likely lidocaine-mediated activation of TRPA1 channels provides an prove beneficial following any surgery of the upper or lower additional pathway for QX-314 entry (Binshtok et al., 2009b). extremity as well as in dental surgical procedures. We also demonstrated that the lidocaine/QX-314-mediated In the present experiments, we set out to systematically effect on mechanical threshold was partially abolished in identify the optimal concentration and ratio of lidocaine and TRPA1 knockout mice (Binshtok et al., 2009b). These findings QX-314 for producing prolonged local analgesia. We found, suggest that the lidocaine/QX-314 effect on mechanical however, that QX-314 when administered alone under inha- threshold is partially mediated via TRPA1 channels. Surpris- lational (isoflurane) anaesthesia begins to produce an effect ingly, the combination of QX-314 and lidocaine produced an on its own at high concentrations (1%, ~35 mM and higher), increase in the duration of the motor block compared with as has been reported previously (Lim et al., 2007). When we lidocaine alone, even though TRPV1 and TRPA1 are not tested administration of QX-314 alone in the absence of the expressed in motor neurons. This may indicate that lidocaine general aesthetic isoflurane, this action disappeared. We con- acts on some other large-pore channel to facilitate QX-314 clude that the TRP activation that has been reported for entry into motor axons. The non-selective block decreased, isoflurane and other general aesthetic agents (Cornett et al., however, at the highest dose of lidocaine used (2%), provid- 2008; Matta et al., 2008; Satoh and Yamakage, 2009), is likely ing the longest ‘pain-specific’ phase (9 h) after the initial sufficient to permit some QX-314 entry into nociceptors short non-selective phase (1 h). This long-lasting differential when administered alone at high enough concentrations, effect may have considerable clinical utility because pain something also reported by other investigators (Ries et al., alone is blocked for 90% of the total time. 2009). What action isoflurane has on motor axons to allow These data clearly suggest therefore, that a combination QX-314 entry needs to be explored. At 0.5% (17 mM) of 0.5% QX-314 and 2% lidocaine may be optimally suited QX-314, we found no effect though, even in the presence of for peripheral nerve block in human patients, offering the isoflurane, and therefore consider this concentration to be a best compromise of long analgesia over short motor block. suitable dose for maximizing selectivity even in the presence Because the duration of perineural lidocaine anaesthesia in of general anaesthetics (Figure S1). rodents (<1 h) is considerably shorter than that found in QX-314, when injected intrathecally in mice at concen- humans (Lemke and Dawson, 2000; Berberich et al., 2009; trations of 5 to 10 mM, has been found to produce marked O’Donnell et al., 2010), it will be interesting to determine if irritation and death in some animals (Schwarz et al., 2010), the lidocaine (2%) QX-314 (0.5%) combination, when something never seen when it is injected subcutaneously or administered in humans, produces an even longer local anal- perineurally at very high doses (Lim et al., 2007; Ries et al., gesic phase than the 9 h seen in the rat. This should be readily 2009). The intrathecal effect of QX-314 administered alone detectable in Phase 1b studies in human volunteers. Whether may represent the action of extracellular QX-314 on some there is a differential change over time in local levels of other target present on central neurons. One known effect of lidocaine and QX-314 in patients because of their different extracellular QX-314 is to block nicotinic ACh receptors. lipid solubility will need to be explored. Conceivably, this could reduce inhibitory synaptic activity in In conclusion, we describe here a preclinical study specifi- the spinal cord, which is enhanced by nicotinic receptor cally designed to translate the strategy of targeting sodium stimulation (Takeda et al., 2003; 2007). In any case, if the channel blockers into nociceptors in a manner applicable for irritant effect of intrathecal QX-314 is duplicated in primates clinical use. The next step, assuming that there are no toxi- it would obviously preclude intrathecal use of QX-314 in cological issues with the local injection of QX-314 in combi- patients; and, to avoid any risk of inadvertent intrathecal nation with lidocaine in peripheral tissue or nerves, will be injection, would also preclude epidural administration. In testing this combination in human volunteers and patients our experience, neither subcutaneous injection nor perineu- to determine the nature, selectivity, depth and duration of ral administration of QX-314 at concentrations up to 2% sensory and motor block. If the clinical data match the pre- (68 mM) even at high volumes produced any observable clinical findings reported here, the combination of lidocaine adverse effects in mice and rats. and its quaternary derivative QX-314 in an injectable formu- Increasing the concentration of lidocaine from 0.5 to lation may be a useful addition for regional pain control, 2.0% markedly increased the duration of analgesia to producing a longer and more selective action than existing mechanical and heat stimuli when combined with 0.5% local aesthetic agents. QX-314. Although lidocaine is used clinically at concentrations up to 4%, it has both a risk of direct neural toxicity (Lirk et al., 2007; Perez-Castro et al., 2009; Werdehausen et al., Conflict of interest and 2009) and systemic CNS/CVS side effects (Dillane and Finu- acknowledgements cane, 2010; Neal et al., 2010), that are particularly evident at higher doses. Moreover, current clinical standards recom- This study was supported by a research grant from Endo mend a lidocaine concentration of 1–2% as optimal for sciatic Pharmaceuticals who have licensed the technology, which

British Journal of Pharmacology (2011) 164 48–58 55 BJP DP Roberson et al. was invented by Bruce Bean and Clifford Woolf, from Dillane D, Tsui BC (2010). From basic concepts to emerging Harvard University and the Massachusetts General Hospital. technologies in regional anesthesia. Curr Opin Anaesthesiol 23: Additional support was obtained from the NIH (C. J. W. and 663–669. B. B., NS039518 and NS064274). We thank David Segal for Enneking F, Wedel D, Horlocker T (2009). The Lower Extremity: technical assistance and Peter Gerner for useful comments Somatic Blockade, 4th edn. Lippincott Williams & Wilkins: and advice. Philadelphia, PA.

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Zhang XF, Shieh CC, Chapman ML, Matulenko MA, Hakeem AH, circles) and pinch tolerance threshold (black squares) pro- Atkinson RN et al. (2010). A-887826 is a structurally novel, potent duced following perisciatic injection of vehicle (0.9% NaCl), and voltage-dependent Na(v)1.8 sodium channel blocker that 1% lidocaine, 2% lidocaine, 0.5% lidocaine N-ethyl bromide attenuates neuropathic tactile allodynia in rats. (QX-314), 1% QX-314, combined doses of 0.5% QX-314 + 1% Neuropharmacology 59: 201–207. lidocaine, 0.5% QX-314 + 2% lidocaine, 1% QX-314 + 1% lidocaine and 1% QX-314 + 2% lidocaine. Supporting information Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied Additional Supporting Information may be found in the by the authors. Any queries (other than missing material) online version of this article: should be directed to the corresponding author for the article. Figure S1 The duration of change in grip strength (blue triangles), thermal (radiant heat, 50°C) response latency (red

58 British Journal of Pharmacology (2011) 164 48–58